MOLECULAR MECHANISMS OF NEURODEGENERATION IN DNA REPAIR DISEASES[unreadable] [unreadable] Cyclopurines as Candidate Neurodegenerative DNA Lesions in Xeroderma Pigmentosum [unreadable] Studies in the past year have continued to make progress in understanding the biological significance of a novel class of oxidative DNA lesions called cyclopurines that are formed in DNA as a result of the hydroxyl radical. These lesions are unique amongst oxidative DNA lesions in that they are specifically repaired by the nucleotide excision repair pathway. We have proposed that the accumulation of cyclopurine lesions on the transcribed strand of active genes is responsible for the neurodegeneration observed in patients with xeroderma pigmentosum who lack the capacity to carry out NER (Brooks, 2006). [unreadable] In addition to blocking transcription, we have also investigated the possibility that RNA polymerase II can bypass cyclopurines in living mammalian cells. For this purpose, we created plasmid DNA constructs in which a single lesion was placed on the transcribed strand of a reporter gene, downstream from an intron. The lesion containing construct, or a lesion-free control, were transfected into NER -deficient cells, and isolated RNA analyzed by RT-PCR followed by restriction digestion and sequencing. Using this approach, we have obtained evidence that two different classes of mutant RNA transcripts are produced when the polymerase bypasses the cyclopurine lesion in vivo. These results represent the first evidence that DNA lesions of the type that are repaired by the NER pathway can stimulate transcriptional mutagenesis. A manuscript describing these results has been submitted and is currently under revision. [unreadable] [unreadable] To better understand the molecular mechanism underlying the formation of mutant transcripts, we have recently adopted a minimal system for studying the effects of cyclopurine (as well as other DNA lesions) on transcription by purified RNA polymerases in vitro. Using the minimal system we have confirmed the blocking effect of the cyclopurine lesion on transcription by both mammalian and bacterial RNA polymerases. Experiments using single rNTPs have shown that the polymerase specifically incorporates U opposite the cyclo-dA lesion, consistent with our in vivo observations. Also, of mechanistic significance, we have found that the effect of the lesion on incorporation is less dramatic than on the extension of an RNA transcript containing a U opposite the lesion, which is also consistent with our in vivo observations. [unreadable] [unreadable] Cyclopurines as Inhibitors of Multiple DNA processing enzymes, and as Structural Probes of DNA Processing Enzyme Functions[unreadable] [unreadable] We have also extended our studies on the inhibition of DNA processing enzymes by cyclopurines. We have found that a single cyclopurine lesion is a complete block to 3 different 5' to 3' exonucleases, and 5 different 3' to 5' exonucleases. Interestingly, we have also identified one bacterial 3' to 5' exonuclease that can completely degrade DNA past the cyclopurine lesion, except when there is a T residue 5' to the cyclo-dA lesion, in which case even this enzyme is blocked. [unreadable] In addition to further emphasizing the unique biological effects of cyclopurine lesions, these studies provide framework for structural approaches to understand, at the atomic level, how cyclopurines exert unique effects. [unreadable] [unreadable] Cyclo-dA and 8-oxo-dA Lesions Stabilize Glycosidic Bonds [unreadable] As noted above, NER is the only know pathway enzymatic pathway for the removal of cyclopurine lesions from DNA. [unreadable] An alternative mechanism for removal of cyclopurines from DNA in the absence of NER might involve spontaneous hydrolysis of the glycosidic bond, followed by removal of the lesion by an AP endonuclease. Several other DNA lesions have been reported to destabilize the glycosidic bonds. However, using in vitro studies, we have now found that the additional 8, 5? bond present in cyclo-dA actually stabilizes the glycosidic bond against hydrolysis. Surprisingly, the glycosidic bond is stabilized to an even greater extant by the structurally less distorting 8-oxo-dA lesion. Thus any mechanism for removal of these lesions that involves spontaneous glycosidic bond hydrolysis is highly unlikely. This work will be submitted for publication shortly. [unreadable] [unreadable] Nuclear Localization of the ATM Protein in Human Purkinje Neurons[unreadable] In addition to our interest in the mechanisms of neurodegeneration in XP, we also maintain in interest in the mechanistic basis of other neurodegenerative conditions associated with hereditary defects in DNA repair, including ataxia telangiectasia (AT) and AT-like disorder (ATLD). AT patients develop a progressive cerebellar degeneration in which cerebellar Purkinje neurons are specifically affected. An understanding of the molecular basis of neurodegeneration in AT has been complicated by published work showing that the ATM (ataxia-telangiectasia mutated) protein, which has been shown in numerous other studies to play a crucial role in the detection of DNA damage, is present in the cytoplasm of Purkinje neurons. These observations indicated a novel role for the ATM protein aside from DNA damage sensing in Purkinje neurons. However, it the past year, we have re-evaluated this question, and found that ATM staining is predominantly localized to the nucleus of human Purkinje neurons. In contrast to other work, our studies utilized postmortem brain tissues from AT patients as negative controls to conclusively establish the specificity of the staining. Ongoing studies are investigating the subnuclear localization of the ATM protein in relation to other proteins, especially components of the MRE11/RAD50/NBS1 complex, in human Purkinje neurons, since mutations in the gene encoding MRE11 result in ATLD.[unreadable] [unreadable] The relevance of these studies derived from the observation that Purkinje neurons of the cerebellum are specifically affected in human alcoholics. Therefore, understanding the mechanisms underlying the specific vulnerability of Purkinje neurons in hereditary repair disease may be of relevance to understanding the mechanisms of Purkinje neuron degeneration in human alcoholics as well.